The overall theme of this Program Project is to elucidate the mechanisms involved in the development of the vertebrate limb. The projects that are proposed involve five separate but closely related topics. They encompass most of the major organ systems and tissue types of the limb, including: the cartilagenous growth region of long bones, the dense connective tissue that comprises tendons, the cellular and molecular components of skeletal muscle, and the endothelial and smooth muscle cells of the vascular system. The Project Leaders, and their associates and collaborators, combine a diversity of backgrounds, skills and technical expertise allowing for broadly-based investigations that require the application of a variety approaches. Experiments will be done at levels ranging from the molecular (e.g., transcriptional regulation), to the cellular (e.g., proliferation and apoptosis), to whole organs (e.g., long bones). The methodologies employed will be equally broad, and will include: molecular isolations and identifications by cDNA cloning and sequencing, manipulations of gene function by transfection, biochemical and immunohistochemical analysis, elctron microscopy, and cell and organ cultures. The first will investigate the growth and differentiation that occurs within the cartilagenous growth region of long bones. The studies will range from the transcriptional regulation of the type X collagen in hypertrophic chondrocytes, to functional analyses of other molecular components upregulated during hypertrophy, to the regulatory functions of the the perichondrium on chondrocyte proliferaton and differentiation. Dr. Sarkar's project will examine the roles of developmentally-expressed isoforms of troponin T, a key component of the Ca++ regulatory myofibrillar protein complex of skeletal muscle. It will also address the mechanism of cytoplasmic RNA-mediated translational control during muscle development . The next project will examine two cellular and molecular mechanisms involved I formation of the vascular system in development: apoptosis of endothelial cells via activation of the Akt family of kinases, and control of smooth muscle during cell proliferation by acquisition of the heparin-responsive phenotype. The last project will also examine the differentiation of skeletal muscle. These studies, however, will focu on the proliferation and survival of myocytes, and how these events are coordinately regulated through the ability of cell cycle activity o modulate the induction of Akt1 and Akt2--protein kinases thought to be involved in survival. Through these various studies we hope to elucidate the mechanisms involved in a variety of aspects of limb develop. We also hope to gain insight into the etiologies of congenital defects in limbs, and in other embryological organ systems.